Longin R-SNARE is retrieved from the plasma membrane by ANTH domain-containing proteins in Arabidopsis

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.

Molecular Mechanisms of Facultative Heterochromatin Formation: An X-Chromosome Perspective

Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox ( Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription ( Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.

Enzymatic C-glycosylation: Insights from the study of a complementary pair of plant O- and C-glucosyltransferases

C-Glycosylation presents a rare mode of sugar attachment to the core structure of natural products and is catalyzed by a special type of Leloir C-glycosyltransferases (C-GTs). Elucidation of mechanistic principles for these glycosyltransferases (GTs) is of fundamental interest, and it could also contribute to the development of new biocatalysts for the synthesis of valuable C-glycosides, potentially serving as analogues of the highly hydrolysis-sensitive O‑glycosides. Enzymatic glucosylation of the natural dihydrochalcone phloretin from UDP‑D-glucose was applied as a model reaction in the study of a structurally and functionally homologous pair of plant glucosyltransferases, where the enzyme from rice (Oryza sativa) was specific for C-glycosylation and the enzyme from pear (Pyrus communis) was specific for O-glycosylation. We show that distinct active-site motifs are used by the two enzymes to differentiate between C- and O-glucosylation of the phloretin acceptor. An enzyme design concept is therefore developed where exchange of active-site motifs results in a reversible switch between C/O-glycosyltransferase (C/O-GT) activity. Mechanistic proposal for enzymatic C-glycosylation involves a single nucleophilic displacement at the glucosyl anomeric carbon, proceeding through an oxocarbenium ion-like transition state. Alternatively, the reaction could be described as Friedel–Crafts-like direct alkylation of the phenolic acceptor.

Activation of an Essential Calcium Signaling Pathway in Saccharomyces cerevisiae by Kch1 and Kch2, Putative Low-Affinity Potassium Transporters

ABSTRACT In the budding yeast Saccharomyces cerevisiae , mating pheromones activate a high-affinity Ca 2+ influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K + and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K + but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K + environments, suggesting they promote K + uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K + currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K + currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K + influx and that HACS may electrochemically respond to K + influx in much the same way as the homologous voltage-gated Ca 2+ channels in most animal cell types.

Identification of chromosomes controlling abscisic acid responsiveness and transcript accumulation of Cor - Lea genes in common wheat seedlings

Abiotic stresses, such as cold, drought or high salinity, seriously affect plant growth and reduce yield in crop species including common wheat (Triticum aestivum L.). The phytohormone ABA plays important roles in plant adaptation to abiotic stress. We compared responsiveness to exogenous ABA, based on root growth inhibition by ABA, among three common wheat cultivars. Seedlings of the cultivars Cheyenne (Cnn) and Hope showed higher ABA responsiveness and higher levels of Cor (cold-responsive)–Lea (late embryogenesis abundant) gene expression than seedlings of Chinese Spring (CS). The chromosomes involved in the regulation of ABA responsiveness and Cor–Lea expression were identified using chromosome substitution lines, in which a chromosome pair of CS was substituted for the corresponding homologous pair of Cnn or Hope. In the CS–Cnn substitution lines, chromosomes 3A, 5A, 5D and 7A increased the ABA responsiveness of CS. Chromosomes 3A and 5A were also involved in the regulation of Cor–Lea gene expression and stomatal response during leaf dehydration. Substitution of CS chromosomes 3A or 5A with the respective homologous pair from Hope also enhanced ABA responsiveness and Cor–Lea expression. In addition, the factors present on chromosomes 4D and 7B of highly responsive cultivars increased Wrab17 expression but had little or no effect on ABA responsiveness. Cultivar differences in ABA responsiveness appear to be determined by genes present on these specific chromosomes in common wheat.

The effect of increased dosage of wheat chromosomes on chromosome pairing and an analysis of the chiasma frequencies of individual wheat bivalents

By crossing the 'Chinese Spring' wheat tetrasomic series with rye, 21 different hybrid genotypes were produced, each with a single different homologous pair of wheat chromosomes. These hybrids enable the effect on chromosome pairing to be assessed for an extra dose of each of the 21 chromosomes. Significant effects were observed with eight of the chromosomes. Chiasma frequencies for each of the 21 pairs of chromosomes were also studied and the differences between the chromosomes were reported. The correlation between chromosome pairing and chiasma formation and chiasma frequency differences between bivalents and genomes are discussed.Key words: wheat, chromosome pairing, chiasma frequencies.

Symmetrical Giant Neurones in Asymmetrical Ganglia: Implications for Evolution of the Nervous System in Pulmonate Molluscs

Two of the largest neurones in the terrestrial snail Achatina fulica are RPrl, located in the right parietal ganglion, and V1, located in the visceral ganglion. Several characteristics of these cells were studied in detail, including morphology, passive and active electrical properties, synaptic inputs, sensory inputs, motor outputs and sensitivity to transmitter substances. The results suggest that RPrl and V1 form a bilaterally homologous pair of cells, yet they reside in asymmetrically placed ganglia. An explanation of this paradox is offered in the proposal that RPrl and V1 were formerly locatedin the bilaterally symmetrical intestinal ganglia. Their contemporary asymmetrical locations are accounted for by the hypothesis that, during the evolution of the pulmonate nervous system, the supraintestinal ganglion fused with the right pallial ganglion and the subintestinal ganglion fused with the visceral ganglion.